Interleukin 4 (IL-4, also known as B cell stimulating factor, or BSF-1) is a cytokine produced by T helper cells, mast cells, and basophils. IL-4 has been shown to possess a broad spectrum of biological activities, including growth co-stimulation of T cells, mast cells, granulocytes, megakaryocytes, and erythrocytes. In addition, IL-4 stimulates the proliferation of several IL-2 and IL-3 dependent cell lines, induces the expression of class II major histocompatibility complex molecules on resting B cells, influences the production of IgE and enhances the secretion of IgE and IgG1 isotypes by lipopolysaccharide-stimulated B cells. IL-4 has been identified to play a critical role in the development of allergic diseases, and is most commonly associated with asthma and allergies; or diseases characterized by difficulty breathing.
IL-4 binds to IL-4 receptor (IL-4R), an endogenous membrane-bound protein on the surface of certain cells. Upon such binding, IL-4R transduces a biological signal to various immune effector cells, thereby triggering a cascade of events that lead to clinical symptoms (Renz H et al., 1991, J Immunol, 146(9):3049-55). Nucleotide and protein sequence determination for IL-4R has been carried out. Mature human IL-4R has three domain structures: an extracellular domain (about 207 amino acids), a membrane passage region (about 24 amino acids), and an intracytoplasmic domain (about 569 amino acids) (European Patent No. EP 585-681 (1994)). Soluble IL-4R (sIL-4R) has also been isolated, cloned and extensively investigated (European Patent No. EP 367-566(1997); Mosley et al., 1989, Cell, 59-335, 1989; U.S. Pat. No. 5,767,065 and Garrone P et al., 1991, Eur J Immunol, 21(6):1365-9). IL-4 preferentially binds to sIL-4R in solution rather than to the endogenous cell-surface IL-4R, thereby preventing cellular activation and blocking the biological response, e.g., the cascade of effects associated with IL-4 and its binding to the endogenous receptor (Renz H et al., 1991, supra. and Renz, H, 1999, Inflamm Res., 48(8): 425-31).
IL4-R has been described as an immunosuppressant and an anti-inflammatory agent, and administration of IL-4R may be beneficial in the treatment of conditions such as allergy, rhinitis, atopic dermatitis, rheumatoid arthritis, graft rejection, chronic graft-versus-host disease (GvH) and systemic lupus erthematosus (SLE) (See, e.g., U.S. Pat. No. 5,856,296; Renz H et al., 1992, J Invest Dermatol, 99(4):403-8; Hackstein H et al., 1999, Tissue Antigens, 54(5):471-7; Rivas D et al., 1995, J Autoimmun, 8(4):587-600; and Schorlemmer H U et al., 1995, Inflamm Res, 44 Suppl 2:S194-6).
Like many biopeptides, IL-4R tends toward instability. It tends to degrade and/or aggregate under extreme conditions (e.g., highly acidic or basic pH, high temperatures) and is susceptible to oxidizing agents and endogenous proteases. The inherent chemical and physical instability of IL-4R makes pharmaceutical formulation particularly problematic. To maintain the stability and bioactivity of the protein, current IL-4R formulations are primarily solution-based, and stored prior to administration as lyophilizates (e.g., U.S. Pat. Nos. 5,856,296; 5,767,065, and 6,063,371). A soluble, solution-based IL-4R peptide composition for administration by inhalation, Nuvance™, is currently in clinical trials for the treatment of asthma (Borish L C et al., 1999, Am J Resp Crit Care Med, 160(6): 1816-23).
Solution-based formulations of IL-4R suffer from drawbacks other than those associated with solution phase instability. First, solution-based formulations take up more room and require more care than solid formulations and, thus, are more costly. Moreover, in general, they must be refrigerated (typically maintained in an environment of 2 to 8° C.) which further restricts the storage and transport options. In addition, many solution-based formulations exhibit a protein concentration loss over time, which is presumably due to the formation of dimers and other protein aggregates in solution. Such formulations frequently must be supplemented with stabilizing additives such as buffers and/or antioxidants to minimize solution instability. Thus, it would be desirable to provide a solid or powder-based composition of IL-4R, particularly one that could not only be stably prepared and stored, but additionally administered in solid form, such as an inhaleable dry powder. Many preclinical and clinical studies with inhaled proteins, peptides, DNA and small molecules have demonstrated efficacy both within the lungs and systemically.
Powder formulations represent an alternative to solution formulations, and proteins, when desired in powder form, are most often prepared as lyophilizates (e.g., U.S. Pat. No. 5,856,296). Unfortunately, lyophilized powders are typically formed as cakes, which require additional grinding and milling, and optionally sieving processing steps to provide flowing powders. In the past few years, spray drying has been employed as an alternative approach for preparing a number of therapeutic protein-based powders, particularly for aerosolized administration (e.g., International Patent Publication Nos. WO 96/32149; WO 95/31479; WO 97/41833, assigned to Inhale Therapeutic Systems, Inc.). Unfortunately, certain proteins, and cytokines in particular, are prone to degradation during spray drying and loss of their secondary structure (Maa, Y. F., et al., J. Pharm. Sciences, 87 (2), 152-159 (1998)). For a representative cytokine, human growth hormone, Mumenthaler reported that spray drying at 90° C. resulted in 4% formation of insoluble aggregates and 21% formation of soluble aggregates—a loss of 25% intact protein (Pharmaceutical Res., 11, 12-20 (1994)). The instability of the illustrative cytokine, hGH, was further demonstrated by Maa, Y. F., et al., ibid, who reported 42% aggregate formation (soluble and insoluble) upon atomization of a solution of hGH.
Additionally, sIL-4R possesses a number of potential instability sites leading to both solution and solid state-based instability. Specifically, sIL-4R contains 7 cysteines (Cys11, 21, 31, 51, 61, 63 and 184), ensuring at least one free sulfhydryl which may be available for intermolecular disulfide linkages. Such intermolecular disulfide linkages lead to the ready formation of dimers, trimers and other self-aggregates. Thus, this molecule is particularly prone to instability. In addition to sites susceptible to aggregation, the IL-4R peptide also has sites susceptible to degradation. For example, sites likely vulnerable to oxidative attack include four methionine residues (Met3, 16, 25, and 67). Additionally, an acid labile Asp-Pro linkage disruptable at low pH is found at amino acid residues 145-146. Two likely deamidation sites include Asn-Gly (26-27), and Asn-Gly (56-57), although the molecule possesses numerous other potential deamidation residues (Asn and Gln).
Thus, the challenge facing the inventors was not only to provide an improved dry powder formulation of IL-4R for overcoming some of the disadvantages associated with solution-based formulations of IL-4R as described above, but also to balance the factors affecting the instability and aerosol properties of IL-4R to arrive at a stable dry powder formulation suitable for pulmonary administration. That is to say, prior to the present invention, the development of a chemically and physically stable, bioactive dry powder of IL-4R that also possesses the physical properties necessary for aerosolization (e.g., high dispersibilities which remain stable over time, appropriate aerodynamic size) was unknown.